Antimicrobial
Properties of Selected Indian Medicinal Plants Against Acne-Inducing Bacteria

*Harisaranraj, R., S. Saravana Babu and Suresh, K.

*Department of Plant Biology and Plant Biotechnology

ChikkaiahNaickerCollege, Erode. (Tamil Nadu) INDIA

*hari83biotech@yahoo.com

Issued: January 01, 2010

Abstract

Propionibacterium acnes and Staphylococcus
epidermidishave been recognized as pus-forming
bacteria triggering an inflammation in acne. The present study was conducted
to evaluate antimicrobial activities of Indian medicinal plants against these
etiologic agents of acne vulgaris. Ethanolic extracts of Rauwolfiaserpentina(roots), Piper nigrum(seeds), Azadirachtaindica (leaves), Cardiospermumhalicacabum (leaves), Momordicacharantia(fruits), Casuarinaequisetifolia (fruits), Cynodondactylon(leaves), Ficusreligiosa(leaves), Euphorbia hirta(roots), Ocimum sanctum (leaves),
Phyllanthusniruri(Whole
plant), and Jasminumsambac(flowers) were tested for
antimicrobial activities by agar well diffusion and broth dilution methods.
The results from the disc diffusion method showed that 07 medicinal plants
could inhibit the growth of Propionibacterium
acnes. Among those Azadirachtaindica, Momordicacharantia, Casuarinaequisetifolia, Rauwolfiaserpentina, Cardiospermumhalicacabum,
Phyllanthusniruri and Piper nigrumhad strong inhibitory
effects. Based on a broth dilution method, the Rauwolfiaserpentinaextract had the greatest antimicrobial effect. Piper nigrumandMomordicacharantia
showed outstanding antimicrobial properties against Propionibacterium
acnes based on the agar well diffusion assay, each had a MIC value of 2.8 and 0.862
mg/ml and a MBC of 2.45 and 0.461 mg/ml for Propionibacterium acnes respectively. In bioautography assay, the Rauwolfiaserpentinaextract produced strong inhibition zones against Propionibacterium acnes. Phytochemical
screening of Rauwolfiaserpentinarevealed the presence of
alkaloid which could be responsible for activity. Taken together, our data
indicated that Rauwolfiaserpentinahad a strong inhibitory effect
on Propionibacterium acnes and Staphylococcus
epidermidis.

Acne vulgaris is a common skin condition, caused by
changes in the pilosebaceous units, skin structures
consisting of a hair follicle and its associated sebaceous gland via androgen
stimulation. It is characterized by non inflammatory follicular papules or comedones and by inflammatory papules, pustules, and
nodules in its more severe forms. Acne vulgaris
affects the areas of skin with the densest population of sebaceous follicles;
these areas include the face, the upper part of the chest, and the back.
Severe acne is inflammatory, but acne can also manifest in non inflammatory
forms. Acne is most common during adolescence, affecting more than 85% of
teenagers, and frequently continues into adulthood. Acne affects areas containing the largest oil glands,
including the face, back, and trunk (Leyden
1997). It is generally characterized by formation of seborrhea, comedone, inflammatory lesions and presence of bacteria Propionibacterium acnes, Staphylococcus epidermidisand Malasseziafurfurin the follicular canal and sebum
production (Leyden 2001). Propionibacterium acnes
have been described as an obligate anaerobic organism. It is implicated in
the development of inflammatory acne by its capability to activate
complements and by its ability to metabolize sebaceous triglycerides into
fatty acids, which chemotactically attract neutrophils. On the contrary, Staphylococcus epidermidis, an aerobic organism,
usually involves in superficial infections within the sebaceous unit
(Burkhart et al., 1999). These
factors provide a potential target for treatment. Propionibacterium
acnes and Staphylococcus epidermidisare
the target sites of antiacne drugs (Leyden 2001 and Hamnerius1996).

Long
term use of antibiotics against acne is outdated because of exacerbated
antibiotic resistance (Degrootet al., 1998 andSwanson
2003). The development of antibiotic resistance is multifactorial,
including the specific nature of the relationship of bacteria to antibiotics,
how the antibacterial is used, host characteristics, and environmental
factors. To overcome the problem of antibiotic resistance, medicinal plants
have been extensively studied as alternative treatments for diseases. In the
present study, 12 medicinal plants, which have been traditionally used as
antimicrobial and anti-inflammatory agents were examined for antimicrobial
activities against microorganisms frequently involved in acne inflammation, Propionibacterium acnes and Staphylococcus epidermidis.

Materials and Methods

Plant material

The
12 plant materials used in this study were collected from various locations
in Tamil nadu, India. The plant was identified
with the Herbarium of Botanical Survey of India, Southern Circle, Coimbatore, India (Accession No. 32561-65)

Microorganisms and media

The
test organisms used in this study were as followed: Propionibacterium
acnes (MTCC 1951) and Staphylococcus
epidermidis (MTCC 931). These bacteria were
obtained from the Microbial Type Culture Collection and Gene Bank, Chandigarh, India All media were purchased
from Himedia.

This
experiment was performed by the method of (Bauer AW et al., 1966) with some
modifications. Propionibacterium acnes was
incubated in brain heart infusion medium (BHI) with 1% glucose for 48 h under
anaerobic conditions and adjusted to yield approximately 1.0×108
CFU/ml. Aliquots of molten BHI with glucose agar were used as the agar base.
A prepared inoculum was added to the molten agar,
mixed, poured over the surface of the agar base and left to solidity. For
agar well diffusion method, a well was prepared in the plates with the help
of a cork-borer (0.85 cm). 100 µl of the test compound was introduced into
the well. The plates were then incubated at 370C for 48 h under
anaerobic conditions in an anaerobic jar (Hi-Media) with gas pack and
indicator strip and the jar was kept in an incubator for 48 h at 37 ± 10C.
Gas packs containing citric acid, sodium carbonate and sodium borohydride were used to maintain and check the anaerobiosis, where citric acid releases carbon dioxide
and sodium borohydride releases hydrogen when they
come in contact with oxygen. An indicator strip of methylene
blue, when introduced into the jar, changes in colour
from white to blue in the absence of anaerobiosis. Staphylococcus epidermidis
was incubated in tryptic soy broth (TSB) for 24 h
at 370C and adjusted to yield approximately 1.0×108 CFU/ml. The
procedures were the same as mentioned above except the plates were incubated
at 370C for 24 h under aerobic conditions. All agar well diffusion
tests were performed in three separate experiments and antibacterial activity
was expressed as the mean of inhibition diameters (mm) (see Table 1).

Determination of minimum inhibitory and
bactericidal concentrations

The
minimal inhibitory concentration (MIC) values were determined by broth
dilution assay (Isao Kubo et al.,
1994, Sahinet
al., 2003 and Kumaret al., 2004). The cultures were
prepared at 24 h and 48 h broth cultures of Staphylococcus epidermidisand Propionibacterium
acnes, respectively. The MIC was defined as the lowest concentration of
the compound to inhibit the growth of microorganisms. 3ml of the Nutrient
yeast glucose broth (NYG) for Propionibacterium
acnes, and Nutrient broth for Staphylococcus epidermidis,
in 10 ml glass screw cap test tube was sterilised
by autoclaving at 1210C for 15min. The medium was cooled and inoculated with
50 μl of the bacterial suspension containing 1
x 108 cells/ml. 1 ml of the plant extracts (100mg/ml) was added to
corresponding test tubes under anaerobic condition. 3 ml of NYG broth
inoculated with 50μl of organisms was taken as positive control. The
test tubes were then kept in anaerobic jar (Hi- Media) with gas pack and
indicator strip and the jar was kept in incubator for 48 h at 37 + 10C. Gas
packs containing citric acid, sodium carbonate and sodium borohydride
were used to maintain and check the anaerobiosis,
where citric acid releases carbon dioxide and sodium borohydride
releases hydrogen when they come in contact with oxygen. An indicator strip
of methylene blue, when introduced into the jar,
changes in colour from white to blue in the absence
of anaerobiosis. For Staphylococcus epidermidis, the test tubes were incubated at 37 ±
10C for 24 h aerobically and growth of Propionibacterium
acnes and Staphylococcus epidermidiswas
measured as a function of turbidity at 660 nm using (Systronics
131) Nephaloturbidometer. The MIC and MBC values of
12 medicinal plant extracts against Propionibacterium
acnes and Staphylococcus epidermidiswere
determined. The results are shown in Table 2 as average values from three
separate experiments per medicinal plants and indicate the susceptibility of
bacteria to the medicinal plant extracts.

Phytochemical screening (Ravishankaraet al., 2002)

The ethanolic extract was subjected to preliminary phytochemical testing for the detection of major chemical
groups. The details of the tests are as follows:

1. Phenols

The ethanolic
extract was spotted on a filter paper. A drop of phoshomolybdic
acid reagent was added to the spot and was exposed to ammonia Vapours (Blue coloration of the spot indicates the presence
of phenols).

2. Braemer’s
test for tannins

To a 2–3 ml of ethanolic
extract, 10% alcoholic ferric chloride solution was added. (Dark blue or
greenish grey coloration of the solution indicate the presence of tannins in
the drug).

3. Liebermann-Burchardt
test for steroids and terpenoids

To 1 ml of ethanolic
extract of drug, 1 ml of chloroform, 2–3 ml of acetic anhydride and 1 to 2
drops of concentrated sulfuric acid were added. (Dark green coloration of the
solution indicate the presence of Steroids and dark pink or red coloration of
the solution indicate the presence of terpenoids).

4. Alkaloids

A drop of ethanolic
extract was spotted on a small piece of precoated
TLC plate and the plate was sprayed with modified Dragendorff’s
reagent. (Orange coloration of the spot indicates the presence of alkaloids).

5. Borntrager’s
test for anthraquinones

About 50 mg of ethanolic
extract was heated with 10% ferric chloride solution and 1 ml of concentrated
hydrochloric acid. The extract was cooled, filtered and the filtrate was
shaken with diethyl ether. The ether extract was further extracted with
strong ammonia. (Pink or deep red coloration of aqueous layer indicate the
presence of anthraquinones).

6. Shinoda test
for flavonoids

To 2–3 ml of ethanolic
extract, a piece of magnesium ribbon and 1 ml of concentrated hydrochloric
acid were added. (Pink red or red coloration of the solution indicate the
presence of flavonoids in the drug).

Bioautography

Bioautography
was performed with bacterial cultures exhibiting high sensitivity to the
extracts. Developed TLC plates were carefully dried for complete removal of
solvent, overlaid with agar containing an aliquot of an overnight culture and
incubated at 37 ◦C. The plates were run in duplicate; one set was used
as the reference chromatogram and the other was used for bioautography.

Results

In the present study, 12 medicinal
plant extracts were examined for antimicrobial activity against Propionibacterium acnes and Staphylococcus
epidermidis. The results showed that 07
extracts could effectively inhibit the growth of Propionibacterium
acnes. Among these, ethanolic extracts of Azadirachtaindica,
Momordicacharantia, Casuarinaequisetifolia, Rauwolfiaserpentina, Cardiospermumhalicacabum,
Phyllanthusniruri and Piper nigrumshowed
strong inhibitory effects (Table 1). Interestingly, Azadirachtaindica, Cardiospermumhalicacabum, MomordicacharantiaandPiper nigrumextracts showed promising
antibacterial activities against both Propionibacteriumacnes and Staphylococcus epidermidis. The
remaining 5 plant extracts had considerable activity against both
microorganisms. Subsequent experiments were conducted to determine
inhibitory concentrations of all selected plant extracts. Rauwolfiaserpentinashowed the potent antimicrobial effect. The MIC and MBC values
against both organisms were more than 5 (Table 2). Further, the plant
extracts was subjected to preliminary Phytochemical
screening for the presence and absence of different chemical groups (Table
3).

and Staphylococcus epidermidis.
The results are shown as mean of triplicate measurements.

Plant
extracts

Susceptibility
of bacteria to medicinal plant extractsa

Propionibacterium acnes

Staphylococcus
epidermidis

MIC
(mg/ml)

MBC
(mg/ml)

MIC
(mg/ml)

MBC
(mg/ml)

Rauwolfiaserpentina

>5

>5

>5

>5

Azadirachtaindica

0.046

0.025

0.265

0.215

Cardiospermumhalicacabum

1.006

1.013

0.524

0.212

Momordicacharantia

0.862

0.799

0.461

0.488

Casuarinaequisetifolia

0.26

0.28

0.325

0.354

Cynodondactylon

0.541

0.365

0.159

0.357

Euphorbia hirta

1.011

0.962

0.451

0.265

Ficusreligiosa

0.538

0.462

0.268

0.164

Ocimum sanctum

1.064

1.068

1.344

0.542

Jasminumsambac

0.132

0.145

0.212

0.253

Phyllanthusniruri

>4

>4

2.1

2.3

Piper nigrum

2.8

2.9

2.45

2.81

Clindamycin

76

80

72

69

aThe results indicate of average of 3 separate experiments

*Clindamycin-
All values are in μg/ml

Discussion

The
similar values of MIC and MBC obtained from this plant against Propionibacterium
acnes suggest that the ethanolic extract of Rauwolfiaserpentinacould
possibly act as a bactericidal agent to this microorganism. In addition, the Cardiospermumhalicacabum extract also showed good
antimicrobial effects against Propionibacterium
acnes with a MIC of 1.006 mg/ml
but a high concentration was required to kill both Propionibacterium
acnes and Staphylococcus epidermidisas
compared to the ethanolic extract of Rauwolfiaserpentina. Piper nigrumandMomordicacharantia
showed outstanding antimicrobial properties against Propionibacterium
acnes based on the agar well diffusion assay, each had a MIC value of 2.8 and 0.862
mg/ml and a MBC of 2.45 and 0.461 mg/ml for Propionibacterium acnes respectively.The plant extracts
were further analyzed by phytochemical screening
for detection of phytoconstituents. The assay for bioautography demonstrated strong inhibition zones of Rauwolfiaserpentinaextract
against the growth of Propionibacterium
acnes.

The
clear zones were located in separate places on the TLC plate, suggesting that
more than one compound possessed an antimicrobial effect. There was no
inhibition zones presented above the bands of the other plant extracts
covered with Propionibacterium acnes. This implied that the
strongest effect of the Rauwolfiaserpentinaextract
was against Propionibacterium acnes. Phytochemical screening of Rauwolfiaserpentinaextract showed positive results for the presence of
alkaloids. Alkaloid and its derivatives have activities against Staphylococcus
aureusand methicillin-resistant
S. aureus
(Valsaraj R et al., 1997). The mechanism of action
of highly aromatic planar quaternary alkaloids such as berberine
and harmane (Hoppet al., 1976) is attributed to their
ability to intercalate with DNA (Phillipsonet al., 1987). It is possible that berberine an alkaloid present in Cosciniumfenestratummay act in the same mechanism to
inhibit Propionibacterium acnes and Staphylococcus
epidermidis. Therefore, the active component of
the Cosciniumfenestratumextract could be of interest for further development as an alternative
treatment for acne.